RESUMEN
MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99mTc-carbonyl [99mTc(H2O)3(CO)3]+ (abbreviated to TcCO) mediated labelling of 99mTc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins.
Asunto(s)
Neoplasias del Colon/diagnóstico por imagen , Neoplasias del Colon/metabolismo , Compuestos de Organotecnecio/farmacocinética , Proteínas Recombinantes de Fusión/farmacocinética , Tecnecio/farmacocinética , gamma-Glutamil Hidrolasa/metabolismo , Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/metabolismo , Animales , Activación Enzimática , Humanos , Inmunoterapia/métodos , Marcaje Isotópico/métodos , Tasa de Depuración Metabólica , Ratones , Especificidad de Órganos , Compuestos de Organotecnecio/química , Profármacos/uso terapéutico , Cintigrafía , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/química , Tecnecio/química , Distribución TisularRESUMEN
New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 degrees C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents.
Asunto(s)
Fragmentos de Inmunoglobulinas/biosíntesis , Mucina-1/inmunología , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Mapeo Epitopo , Femenino , Humanos , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/inmunología , Ratones , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
Multimerization of antibody fragments increases the valency and the molecular weight, both identified as key features in the design of the optimal targeting molecule. Here, we report the construction of mono-, di-, and tetrameric variants of the anti-tumor p185(HER-2) single chain Fv fragment 4D5 by fusion of self-associating peptides to the carboxyl terminus. Dimeric miniantibodies with a synthetic helix-turn-helix domain and tetrameric ones with the multimerization domain of the human p53 protein were produced in functional form in the periplasm of Escherichia coli. We have directly compared these molecules and the single-chain Fv fragment in the targeting of SK-OV-3 xenografts. Tetramerization of the 4D5 antibody fragment resulted in increased serum persistence, significantly reduced off-rate, due to the avidity effect, both in surface plasmon resonance measurements on purified p185(HER-2) and on SK-OV-3 cells. The (99m)technetium-tricarbonyl-labeled tetrameric 4D5-p53 miniantibody localized with the highest dose at the tumor and remained stably bound for at least 72 h. The highest total dose was 4.3% injected dose/g after 24 h, whereas the highest tumor-to-blood ratio was found to be 13.5:1 after 48 h, with a total dose of 3.2% injected dose/g. The tetramer shows no higher avidity than the dimer, presumably since the simultaneous binding to more than two antigen molecules on the surface of cells is not possible, and the improvement in performance over the dimer must at least be due in part to the molecular weight. These results demonstrate that multimerization by self-associating peptides can be used for the development of more effective targeting molecules for medical diagnostics and therapy.
Asunto(s)
Anticuerpos/inmunología , Péptidos/metabolismo , Receptor ErbB-2/química , Receptor ErbB-2/inmunología , Animales , Anticuerpos/metabolismo , Cromatografía en Gel , Clonación Molecular , Dimerización , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Femenino , Humanos , Cinética , Ratones , Ratones Endogámicos BALB C , Modelos Genéticos , Periplasma/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Radioinmunoensayo , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/metabolismo , Tecnecio/farmacocinética , Temperatura , Factores de Tiempo , Distribución Tisular , Células Tumorales CultivadasRESUMEN
UNLABELLED: An 131I labeled trivalent antigen binding construct, formed from 3 Fab' fragments of murine anti-CEA monoclonal antibody (Mab) 35, has shown favorable biokinetics in animal studies. OBJECTIVES: The aim of this study was to evaluate biodistribution and tumor uptake of 131I-F(ab')3 in patients and its potential utility for radioimmunotherapy of CEA expressing tumors. PATIENTS AND METHODS: Six patients (5 M, 1 F; age 62 +/- 13 y) with liver metastases of colorectal cancer, scheduled for hepatic surgery were studied by 2-3 whole body scans immediately post infusion of 111-137 MBq of 131I labeled Mab 35 F(ab')3 and up to 72 h. Circulating CEA ranged from 1.2 to 1930 ng/ml. We evaluated plasma and whole body clearance, activity accumulation by post-surgical ex-vivo tissue measurement in primary tumor (T) and metastases (M), and calculated M to blood (M/B) and M to liver (M/L) ratios. RESULTS: All known tumor sites were detected by immunoscintigraphy and confirmed at surgery. Whole body effective T1/2 calculated in two patients was 51.5 h and 55.6 h respectively. Effective serum T1/2 was mono-exponential in 3 patients (short observation interval) with 20.9 +/- 7 h and bi-exponential in three with alpha T1/2 of 6.3 +/- 1 h and beta T1/2 of 38.6 +/- 5 h. In a patient with concomitant colic and hepatic lesions uptake of primary tumor was 0.0071% injected dose per gram of tissue (%ID/g) and mean metastases activity was 0.0275 %ID/g at 48 h. In the 3 patients who had surgery at 48 h, mean uptake in metastases and normal liver was 0.0182 %ID/g and 0.0021 %ID/g, respectively (M/L 8.67). In the single subject followed until 7 days post infusion, residual activity in liver metastases was 10 times higher than in normal parenchyma. CONCLUSIONS: Tumor uptake and tumor to blood ratio, as well as serum clearance of the triconstruct are similar to those observed with intact iodinated anti-CEA antibodies. In the patient studied for 7 days the tumor residence time was favorable. Further improvements, however, need to be obtained before considering this approach for radioimmunotherapy.
Asunto(s)
Adenocarcinoma/secundario , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Antineoplásicos/uso terapéutico , Antígenos de Neoplasias/inmunología , Antígeno Carcinoembrionario/inmunología , Neoplasias Colorrectales/radioterapia , Inmunoconjugados/uso terapéutico , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Radioisótopos de Yodo/farmacocinética , Neoplasias Hepáticas/secundario , Radioinmunodetección , Radioinmunoterapia , Adenocarcinoma/sangre , Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/inmunología , Adenocarcinoma/radioterapia , Adenocarcinoma/cirugía , Anciano , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos/sangre , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/diagnóstico por imagen , Neoplasias Colorrectales/inmunología , Terapia Combinada , Femenino , Semivida , Humanos , Fragmentos Fab de Inmunoglobulinas/sangre , Radioisótopos de Yodo/sangre , Radioisótopos de Yodo/uso terapéutico , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/cirugía , Masculino , Tasa de Depuración Metabólica , Ratones , Persona de Mediana Edad , Distribución TisularRESUMEN
Two new aporphinoid alkaloids, f1ttowianthine and 11-methoxylettowianthine were isolated from the root bark of Lettowianthus stellatus, together with the new sesquiterpene 11-hydroxyguaia-4,6-diene and the known compounds liriodenine, (Z)-7-octadecen-9-ynoic acid, methyl (2E,6E,10R)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate, methyl (2E,6E,10R)-10,11-dihydroxy-3,7,11-trimethyl-2,6-dodecadienoate , and 3,4,5-trimethoxyphenol. The structure elucidation was achieved by spectroscopic methods.
Asunto(s)
Alcaloides/aislamiento & purificación , Aporfinas , Sesquiterpenos/aislamiento & purificación , Árboles/química , Alcaloides/química , Alcaloides/farmacología , Animales , Antimaláricos/química , Antimaláricos/aislamiento & purificación , Antimaláricos/farmacología , Cromatografía en Capa Delgada , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Raíces de Plantas/química , Plasmodium falciparum/efectos de los fármacos , Sesquiterpenos/químicaRESUMEN
The epithelial glycoprotein-2 is abundantly expressed on many solid tumors and is a suitable target for antibody-based therapy. In the present study, an antiepithelial glycoprotein-2 single-chain Fv (scFv) was derived from the hybridoma MOC31 by phage display. Despite its high affinity (KD = 3.9 x 10(-9) M), however, this antibody fragment failed to significantly enrich at lung tumor xenografts in mice, mostly because of its insufficient thermal stability. To overcome this limitation, the antigen-binding residues of the MOC31 scFv fragment were grafted onto the framework of the highly stable and well-folding anti-c-erbB2 scFv 4D5. Further modification of the resulting 4D5 MOC-A, which was performed by transferring eight additional residues of the heavy chain variable domain core of the parent MOC31 antibody, produced 4D5 MOC-B, resulting in increased serum stability at 37 degrees C and also significantly improved expression behavior while retaining the antigen specificity and affinity of the parent MOC31 scFv. In mice, the scFv 4D5 MOC-B, which was radiolabeled with 99mtechnetium using a new histidine-tag specific labeling method (Waibel et al., Nature Biotechnol., 17: 897-901, 1999), showed favorable blood clearance and efficient enriches at lung tumor xenografts, with a tumor:blood ratio of 5.25 and a total dose of 1.47% injected dose per gram after 24 h. Biophysical properties such as high thermal stability are thus decisive for whether these molecules are useful in vivo, and our approach may provide a general strategy to solve this problem. This is also the first report of using a humanized anti-EGP-2 scFv in vivo for targeting solid tumors, which is a promising targeting moiety for the diagnostics and therapy of EGP-2-positive tumors in patients.
Asunto(s)
Anticuerpos Antineoplásicos/química , Antígenos de Neoplasias/inmunología , Moléculas de Adhesión Celular/inmunología , Calor , Fragmentos de Inmunoglobulinas/química , Región Variable de Inmunoglobulina/química , Receptor ErbB-2/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antineoplásicos/aislamiento & purificación , Anticuerpos Antineoplásicos/metabolismo , Especificidad de Anticuerpos , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Molécula de Adhesión Celular Epitelial , Femenino , Humanos , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Fragmentos de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/metabolismo , Marcaje Isotópico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Químicos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Receptor ErbB-2/metabolismo , Alineación de Secuencia , Tecnecio , Células Tumorales CultivadasRESUMEN
UNLABELLED: A new peptide labeling method that uses the organometallic aquaion [99mTc(H2O)3(CO)3]+ has been developed. METHODS: A selection of amino acids was labeled at different concentrations with the organometallic aquaion, and the labeling yield was determined by high-performance liquid chromatography. This investigation has shown histidine to be a very potent ligand, with specific activities of up to 6 TBq/micromol (160 Ci/micromol) ligand. Histidine derivatives have been coupled to neurotensin(8-13) (NT[8-13]) and have been labeled with the aquaion, resulting in high specific activities with (N(alpha)-histidinyl)acetic acid-NT(8-13) similar to those with histidine. RESULTS: Histidine derivatives of NT(8-13) labeled using this approach fully retained their receptor affinity, showing KD values of all investigated NT analogs below 1 nmol/L on colon carcinoma HT29 cells. Biodistrbution experiments in BALB/c mice showed complete clearance of (N(alpha)-histidinyl)acetic acid-NT(8-13) from the blood after 24 h and no unwanted accumulation in any tissue. CONCLUSION: The novel labeling method using the organometallic 99mTc-aquaion combines the advantage of highest specific activities with minimal functionalization of proteins and peptides under retention of biologic affinity.
Asunto(s)
Péptidos , Tecnecio , Animales , Cromatografía Líquida de Alta Presión , Histidina , Humanos , Marcaje Isotópico , Ratones , Ratones Endogámicos BALB C , Neurotensina , Radiofármacos , Distribución TisularRESUMEN
We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.
Asunto(s)
Histidina/química , Marcaje Isotópico/métodos , Compuestos de Organotecnecio/síntesis química , Radiofármacos/síntesis química , Proteínas Recombinantes/química , Tecnecio , Aldehídos , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Cromatografía de Afinidad , Humanos , Fragmentos de Inmunoglobulinas/química , Cetonas , Ratones , Ratones Desnudos , Mucina-1/inmunología , Compuestos de Organotecnecio/farmacocinética , Radiofármacos/farmacocinética , Proteínas Recombinantes/farmacocinética , Distribución TisularRESUMEN
Immunoprecipitation after cell surface labeling of human neuroblastoma cells showed that the anti-neuroblastoma monoclonal antibody (mAb) chCE7 binds to a 200,000 M(r) cell surface protein. The protein was partially purified by immuno-affinity chromatography from a human renal carcinoma and a human neuroblastoma cell line, which both showed high levels of binding of MAb chCE7. NH(2)-terminal sequences of 18 and 15 amino acid residues were determined. Both sequences isolated from the renal carcinoma and the neuroblastoma cells showed strong homology to human cell adhesion molecule L1 (L1-CAM), and both were characterized by the NH(2)-terminal deletion of 5 amino acids, comprising exon 2 of L1-CAM. Reverse trancription-polymerase chain reaction (RT-PCR) analysis of the regions spanning exon 2 and exon 27 of L1-CAM indicated that in neuroblastoma cells both transcripts for the full-length and exon-deleted forms are present, whereas in the renal carcinoma cell lines only the exon-deleted L1-CAM isoform were detected. Western blot analysis showed that 6 of 7 tested renal carcinoma cell lines and 5 of 15 renal carcinoma tissues expressed L1-CAM. In normal adult kidney tissue, very low levels of protein expression were found. Northern blot analysis confirmed that in renal carcinoma and neuroblastoma cell lines L1-CAM mRNA levels are correlated with protein expression.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Neoplasias Renales/química , Glicoproteínas de Membrana/análisis , Proteínas de Neoplasias/análisis , Moléculas de Adhesión de Célula Nerviosa/análisis , Neuroblastoma/química , Animales , Células CHO , Cricetinae , Humanos , Riñón/química , Complejo de Antígeno L1 de Leucocito , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/inmunología , Pruebas de Precipitina , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
The basic amino acid L-lysine was administered to mice in an attempt to circumvent unwanted renal accumulation of 67Cu-labelled F(ab')2 fragments derived from the anti-NCAM IgG1, SEN7 and anti-CEA IgG1 monoclonal antibody (MAb)35. In control experiments, significant renal uptake of both 67Cu-labelled F(ab')2 fragments was observed, radiolabel being primarily localised to proximal tubules in the renal cortex. Following optimised L-lysine dosing protocols, renal uptake of 67Cu-MAb35 F(ab')2 was inhibited by up to 42%. Surprisingly, little inhibition (< 10%) of 67Cu-SEN7 F(ab')2 uptake was observed. Experiments to investigate this differential inhibition indicated that inhibition of MAb35 F(ab')2 uptake was relatively short-lived (approx. 6 hr), whilst no apparent differences were found in blood clearance rates between either 67Cu-F(ab')2 fragment. L-lysine administration caused a significant diuresis with high levels of intact 67Cu-labelled SEN7 and MAb35 F(ab')2 appearing in the urine, possibly due to blockade of renal uptake and lysine-induced increases in glomerular membrane permeability. Iso-electric focusing studies failed to identify any charge differences between the 67Cu-labelled F(ab')2 fragments, although a cathodal migration of all 67Cu-labelled samples, presumably due to the net positive charge conferred by addition of 67Cu2+ ions, was observed. Our results demonstrate that in addition to net charge, other unidentified characteristics may influence renal accumulation of radiometal-labelled F(ab')2 fragments and their inhibition by L-lysine.
Asunto(s)
Radioisótopos de Cobre , Fragmentos Fab de Inmunoglobulinas/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Lisina/farmacología , Animales , Anticuerpos Monoclonales/metabolismo , Antígeno Carcinoembrionario/inmunología , Femenino , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fab de Inmunoglobulinas/orina , Inmunoglobulina G/metabolismo , Punto Isoeléctrico , Marcaje Isotópico , Cinética , Ratones , Ratones Endogámicos ICR , Moléculas de Adhesión de Célula Nerviosa/inmunologíaRESUMEN
Microsomal long-chain acyl-CoA synthetase (EC 6.1.2.3.) has been suggested to be involved in the stereoselective formation of the CoA thioester of ibuprofen. In this study, we demonstrated that the microsomal enzyme from rat liver responsible for palmitoyl-CoA synthesis also catalyzes the formation of R-ibuprofenoyl-CoA in a Mg(2+)- and ATP-dependent process. Long-chain acyl-CoA synthetase from rat liver microsomes was purified to homogeneity as evidenced by SDS-gel electrophoresis. Simultaneous measurements of palmitoyl-CoA and R-ibuprofenoyl-CoA formation with HPLC in various fractions and purification steps during protein isolation revealed a high correlation between both activities. The purification procedure included solubilization of the microsomes obtained from rat livers with Triton X-100 and subsequent chromatography of the 100,000 x g supernatant on blue-sepharose, hydroxyapatite, and phosphocellulose. The purified enzyme exhibited an apparent molecular weight of 72 kDa as estimated by SDS gel electrophoresis, with specific activities of 71 nmol.min-1.mg-1 protein and 901 nmol.min-1.mg-1 protein for formation of R-ibuprofenoyl-CoA and palmitoyl-CoA, respectively. Palmitoyl-CoA formation catalyzed by the purified enzyme exhibited biphasic kinetics indicative of two isoforms, a high-affinity (KM 0.13 +/- 0.11 microM), low-capacity form and a low-affinity (KM 81 +/- 11.5 microM), high-capacity form. In contrast, measurement of R-ibuprofenoyl-CoA synthesis over a concentration range from 5 to 3000 microM showed the participation of a single CoA ligase with a KM of 184 +/- 19 microM, corresponding to the low-affinity isoform of palmitoyl-CoA synthesis with a marked enantioselectivity towards the R-form of ibuprofen. R-ibuprofenoyl-CoA formation of the enzyme preparation was inhibited by palmitic acid (KI 13.5 +/- 0.5 microM) and S-ibuprofen (KI 405 +/- 10 microM). In summary, these data give strong evidence for the identity of R-ibuprofenoyl-CoA and long-chain acyl-CoA synthetase.
Asunto(s)
Coenzima A Ligasas/metabolismo , Ibuprofeno/metabolismo , Hígado/metabolismo , Microsomas/metabolismo , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Animales , Relación Dosis-Respuesta a Droga , Electroforesis , Masculino , Ácidos Palmíticos/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Steady-state pharmacokinetics of morphine and morphine-6-glucuronide (M-6-G) after intravenous administration of either morphine or M-6-G were determined in healthy volunteers. With a dosing regimen calculated on the basis of data obtained in a first series of experiments in four subjects (morphine: intravenous loading dose of 0.24 mg/kg for 5 minutes and an intravenous infusion of 0.069 mg.kg-1.hr-1 for 4 hours; M-6-G: loading dose of 0.011 mg/kg for 5 minutes and an infusion of 0.006 mg.kg-1.hr-1 for 4 hours), it was possible to yield plasma concentrations of morphine and M-6-G in another four subjects close to predefined targeted levels (35 and 45.5 ng/ml morphine and M-6-G, respectively). This dosing regimen may be used in further pharmacodynamic studies to compare the analgesic effects of morphine and M-6-G. In addition, metabolite kinetics of M-6-G were calculated as a function of time with use of a linear systems approach to the estimation of rate and fraction of morphine glucuronidation to M-6-G.
Asunto(s)
Analgésicos Opioides/farmacocinética , Derivados de la Morfina/farmacocinética , Morfina/farmacocinética , Adulto , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/sangre , Cromatografía Líquida de Alta Presión , Esquema de Medicación , Humanos , Infusiones Intravenosas , Modelos Lineales , Masculino , Morfina/administración & dosificación , Morfina/sangre , Derivados de la Morfina/administración & dosificación , Derivados de la Morfina/sangre , Valores de ReferenciaRESUMEN
Human CD24 is a highly glycosylated glycosylphosphatidylinositol-linked (GPI-linked) cell surface protein. GPI-linked proteins are involved in signal transduction mediated by members of the protein tyrosine kinase (PTK) family. Therefore we studied associated molecules providing the signaling capacity of CD24. Lysates of SW2 and K562 cells were analysed for expression of PTK of the c-src family by Western blotting. We identified c-fgr in SW2 lysates and c-fgr and also lyn in K562 lysates. To study a putative association of these PTK with CD24 we performed immunoprecipitations with the mAb SWA11 directed against CD24. Western analysis of the precipitates showed an association of c-fgr with CD24 in SW2 cells and lyn in K562 cells. We conclude that either c-fgr or lyn is physically associated with CD24 in a cell-type depending manner. An involvement of these complexes in signaling phenomenons of CD24 in small cell lung cancer and in leukaemias is discussed.
Asunto(s)
Antígenos CD/metabolismo , Carcinoma de Células Pequeñas/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Glicoproteínas de Membrana , Proteínas Proto-Oncogénicas/metabolismo , Familia-src Quinasas/metabolismo , Antígeno CD24 , Carcinoma de Células Pequeñas/enzimología , Humanos , Leucemia Eritroblástica Aguda/enzimología , Pruebas de Precipitina , Transducción de Señal , Células Tumorales CultivadasRESUMEN
A correlation between CD24 expression and higher intrinsic radiation sensitivity has been described in B-lineage acute lymphoblastic leukaemia (B-ALL). We recently identified the SCLC surface antigen Cluster-4 (CL-4) to be identical to the B cell differentiation marker CD24, except for one amino acid residue. The CD24/CL-4 antigen is highly expressed on SCLC, but rarely on NSCLC cells. In order to investigate the influence of the expression of CD24/CL-4 on the radiation sensitivity in a non-leukaemic cell system, sublines of the human SCLC H249 cell line transfected with mutated ras oncogene, and differing in their CD24/CL-4 expression, were studied. In addition, we stably transfected the NSCLC A125 cell line and the mouse fibroblast NIH3T3 cell line with the CL-4 cDNA. The differential expression of CD24/CL-4 on the cells had no influence on morphology, proliferation and cloning efficiency. Radiation studies were done with cells in exponential growth phase. In the highly resistant NSCLC A125 cells no difference in radioresponsiveness was observed between CD24/CL-4 expressing and non-expressing cells. In the rather radiosensitive cells, similar responses to radiation were observed between CD24/CL-4 expressing and non-expressing SCLC H249-ras cells, whereas the CL-4 transfected NIH3T3 mouse fibroblasts showed a substantially higher radioresistance than the CD24/CL-4 non-expressing control cells. In conclusion, the correlation between CD24/CL-4 expression and radiation sensitivity is controversial and depends on the cell type.
Asunto(s)
Antígenos CD/metabolismo , Carcinoma de Células Pequeñas/fisiopatología , Glicoproteínas de Membrana , Células 3T3 , Animales , Antígeno CD24 , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , Técnicas In Vitro , Ratones , Tolerancia a Radiación , Transfección , Células Tumorales CultivadasRESUMEN
Cluster-4 and CD24 cDNA's have recently been cloned from the small cell lung carcinoma (SCLC) cell line SW2 and from the erythroleukemia cell line K562, respectively. The only difference in the coding sequence, between cluster-4 and CD24 antigens is the substitution of a single base pair leading to a substitution of Val by Ala near the putative glycosylphosphatidylinositol (GPI) anchorage sites of the mature protein. Here we demonstrate that the nucleotide substitution which distinguishes the cluster-4 and CD24 antigen genes is due to an allelic polymorphism on chromosome band 6q21. In addition, we identified by Southern blotting and PCR of DNA from somatic human x hamster hybrid cell lines homologues of cluster-4/CD24 on the Y chromosome and chromosome 15. We suggest, however, that the gene on 6q21 is the active locus since the mRNA of cell lines always represents the allelic variants found on chromosome 6. The distribution pattern of this allelic polymorphism in SCLC cell lines and leukocytes of healthy donors did not reveal any obvious relationship with disease. However, it is noteworthy that homozygosity for cluster-4 was found in only one case whereas heterozygosity and homozygosity for CD24 both contribute up to 50% of the samples examined.
Asunto(s)
Antígenos CD/genética , Carcinoma de Células Pequeñas/genética , Cromosomas Humanos Par 6 , Neoplasias Pulmonares/genética , Glicoproteínas de Membrana , Alelos , Secuencia de Bases , Antígeno CD24 , Mapeo Cromosómico , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Células Tumorales CultivadasRESUMEN
Coenzyme A thioester formation is reported to be the first step of chiral inversion of R-ibuprofen. In order to investigate the mechanism of this reaction adenylate derivatives of the ibuprofen enantiomers were synthesized chemically. R- and S-ibuprofenyl-adenylates as well as free acids were incubated with rat liver mitochondria in the presence of coenzyme A, MgCl2 with or without ATP. The optical antipodes formed by inversion and the coenzyme A thioester derivatives of both enantiomers were found after incubation of both R- or S-ibuprofenyl-adenylate and R-ibuprofen. By contrast, after incubation with S-ibuprofen neither R-enantiomer nor coenzyme A thioesters were detected. These experiments suggest that the formation of R-ibuprofenyl-adenylate may be the first stereoselective step of chiral inversion.
Asunto(s)
Ibuprofeno/metabolismo , Animales , Coenzima A/metabolismo , Ésteres , Ibuprofeno/química , Técnicas In Vitro , Masculino , Mitocondrias Hepáticas/metabolismo , Ratas , Ratas Sprague-Dawley , EstereoisomerismoRESUMEN
The intact anti-SCLC monoclonal antibody (MAb) SEN7 and its F(ab')2 were labelled with the beta-emitting isotope 67Cu. Both materials retained their biological activity in vitro as determined by the Lindmo assay. In a direct comparison of in vivo distribution in a xenograph model, 131I- and 67Cu-labelled intact SEN7 showed similar absolute tumour accumulation. Blood levels were markedly lower in the case of the 67Cu-labelled antibody, resulting in improved tumour:blood ratios which reached a maximum of 13:1 compared with only 4.5:1 for 131I-SEN7. In the case of the 67Cu-labelled F(ab')2, very high accumulation of the nuclide was observed in the kidney. Levels of radio copper in liver and spleen were also found to be significantly raised when compared with radio iodine. SWA20, a MAb which had previously failed to show any selective in vivo accumulation in tumour xenografts when labelled with radio iodine showed higher and more stable tumour accumulation when labelled with 67Cu.
Asunto(s)
Anticuerpos Monoclonales , Carcinoma de Células Pequeñas/diagnóstico por imagen , Radioisótopos de Cobre/farmacocinética , Radioisótopos de Yodo/farmacocinética , Neoplasias Pulmonares/diagnóstico por imagen , Radioinmunodetección/métodos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/metabolismo , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/metabolismo , Indicadores y Reactivos , Ratones/inmunología , Ratones Desnudos , Factores de Tiempo , Distribución Tisular , Trasplante HeterólogoRESUMEN
The present study describes a comparison of two potent immunotoxins which utilise an identical targeting component, a monoclonal antibody (SEN7) specific for small cell lung cancer (SCLC), conjugated to two different effector components, blocked ricin (bR) and Pseudomonas exotoxin A (PE). SEN7 recognises a novel epitope on the neural cell adhesion molecule (NCAM) which is highly associated with SCLC. The immunotoxins SEN7-PE and SEN7-bR were selectively and potently active against a number of SCLC cell lines, of both classic and variant morphologies, inhibiting the incorporation of [3H]leucine with IC50 values ranging between 22 pM and 85 pM and between 7 pM and 62 pM for SEN7-PE and SEN7-bR respectively. Intoxication by both immunotoxins proceeded rapidly following short 2 h lag phases; the initial rates of protein synthesis inhibition occurred with t50 values of 6.5 h for SEN7-PE and 5.5 h for SEN7-bR. Monensin drastically enhanced the cytotoxic activity of the weakly active SEN7-ricin A-chain by 2,100-fold and of SEN7-bR by 80-fold but had no effect on SEN7-PE. In limiting dilution assays, four and more than 4.5 logs of clonogenic SW2 tumour cells were selectively eliminated from the cultures during continuous exposure to the immunotoxins SEN7-PE and SEN7-bR respectively, while antigen-negative cells required up to 1,000-fold more drug for a similar cell kill. SW2 cells surviving SEN7-bR treatment in the cultures did not express NCAM and consequently were not selectively killed by SEN7 immunotoxins. SW2 cells surviving continuous exposure to SEN7-PE showed no alteration in NCAM expression but were more resistant to intoxication mediated by PE. These cells were still sensitive to SEN7-bR.
Asunto(s)
ADP Ribosa Transferasas , Toxinas Bacterianas , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/inmunología , Moléculas de Adhesión Celular Neuronal/inmunología , Epítopos/inmunología , Exotoxinas/toxicidad , Inmunotoxinas/inmunología , Inmunotoxinas/toxicidad , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Ricina/toxicidad , Factores de Virulencia , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Carcinoma de Células Pequeñas/metabolismo , Técnicas de Cultivo , Sinergismo Farmacológico , Humanos , Inmunotoxinas/metabolismo , Cinética , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/biosíntesis , Células Tumorales Cultivadas , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Expression of isoforms of the CD44 hyaluronan receptor/lymph-node endothelial receptor by human tumour cells is thought to play a role in tumour growth and metastasis. These isoforms which vary in the length of the extracellular domain are generated by differential RNA splicing that involves the 10 alternative exons (v1 to v10) encoding the membrane proximal region of the molecule. Several tumours have been shown to over-express CD44 containing the v6 exon, and this, together with other evidence, has led to the suggestion that v6 may play a causative role in tumour metastasis. In this report we have compared the expression of CD44 isoforms between different lung tumour lines, including SCLC, squamous-cell carcinoma, adenocarcinoma and mesothelioma, using both RT-PCR and fluorescent antibody staining with a panel of CD44 exon-specific monoclonal antibodies (MAbs). Our results show large differences in vCD44 expression between individual tumour lines. Little or no vCD44 containing the metastasis-associated v6 exon was detected in most tumours, including the highly metastatic SCLC lines. Indeed, the SCLC lines and some squamous-cell carcinomas contained only very low levels of either vCD44 or CD44H, indicating that CD44 expression may not always correlate with tumour development or dissemination. One of the squamous-cell carcinomas studied (HOTZ) was found to express a complex mixture of CD44 splice variants similar to the immortalized normal bronchial epithelial line BEAS-2B. Cloning and sequencing of vCD44 from the HOTZ cell line yielded several splice variants that have also been identified on leukaemic cells, normal keratinocytes and activated peripheral-blood lymphocytes.
Asunto(s)
Empalme Alternativo , Carcinoma de Células Pequeñas/metabolismo , Proteínas Portadoras/biosíntesis , Neoplasias Pulmonares/metabolismo , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/biosíntesis , Receptores Mensajeros de Linfocitos/biosíntesis , Secuencia de Bases , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proteínas Portadoras/genética , Línea Celular , Células Clonales , Clonación Molecular , Cartilla de ADN , Citometría de Flujo , Expresión Génica , Humanos , Receptores de Hialuranos , Neoplasias Pulmonares/genética , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Receptores de Superficie Celular/genética , Receptores Mensajeros de Linfocitos/genética , Células Tumorales CultivadasRESUMEN
We have investigated immunologically the molecular association of the cell-surface sialoglycoprotein antigens cluster-5 (CL-5) and cluster-5A (CL-5A), known to be co-expressed in human small-cell lung cancer (SCLC). CL-5 antigen is exclusively defined by IgM antibodies as represented by MAb LAM8, whereas CL-5A antigen is exclusively defined by IgG antibodies as represented by MAbs SWA20 and SEN31. Because of the unavailability of purified antigens, the question of the molecular relationship between these antigens was addressed by immunological studies. We generated an anti-anti-idiotypic MAb of the IgG isotype using a CL-5-antigen-mimicking anti-idiotype defined by rat MAb Ly8-229 as an immunogen to circumvent the avidity problems observed with the IgM MAb LAM8 in binding-competition experiments. In addition, we developed a heterologous double antibody sandwich assay able to identify circulating CL-5/5A antigens in pre-treatment sera of patients with SCLC. The results of both types of immunological studies demonstrated the expression of CL-5 and CL-5A antigens on a single molecule, both in cellular assays and in assays detecting antigens shed into the serum of patients with SCLC.